The virus that causes AIDS, the human immunodeficiency virus HIV is believed to be one of the major threats to human life and health worldwide. Even back in 1988 an article in Scientific American by J. M. Mann, J. Chin, P. Piot and T. Quinn estimated that more than a quarter of a million AIDS cases had occurred in the U.S.A. up to then and that 5-10 million people were infected worldwide. An article in the same magazine ten years later “Defeating Aids: What will it take? (July 1998 page 62) revealed that worldwide 40 million people had contracted HIV and almost 12 million had died leaving over 8 million orphans. During 1997 alone nearly 6 million people acquired HIV and some 23 million perished including 460,000 children.
Although 90% of HIV infected people live in developing countries well over 90% of money for care and prevention is spent in industrial countries. The very expensive triple therapy drugs (over US$10,000-$15,000 per person per year) are well beyond the reach of individuals in developing countries in sub Saharan Africa and Asia. In 1999 alone, 300,000 people died in Ethiopia from AIDS far exceeding deaths from famine (12 Apr. 2000, The Irish Examiner). Up to a quarter of South Africa's non-whites currently face death from AIDS in the next ten years (11 May 2000, The Irish Examiner, by G. Dyer). There is thus a desperate need for cheap, easily made and efficient anti-HIV agents for the developing world.
The HIV has been studied more intensively than any other virus and we now have a general picture of how the genes and proteins in the HIV virus particle operate, although we don't have a clear understanding of what controls the replication and how it destroys the human immune system. There are in fact many strains of HIV. The two main ones are HIV-1 and HIV-2. HIV-2 is prevalent in West Africa and produces a less severe disease than does HIV-1 the most common form elsewhere.
The life cycle of the virus is described below in some detail since for a drug to be effective it has to interfere with at least one stage of its life cycle. The HIV virus particle is roughly spherical shaped and is about a thousandth of a millimeter across. Its outer membrane consists of lipid molecules which possess many viral protein spikes projecting outwards. Each spike is thought to consist of four molecules of glycoprotein gp120 with the same number of glycoprotein gp41 molecules embedded in the membrane itself. These envelope proteins come into play when HIV binds and then enters target cells. Gp120 can bind tightly to CD4 proteins sited in the membranes of immune system cells especially T lymphocytes also called T cells. This is the first stage of the infection which is followed by fusion of the virus and T cell membrane, a process governed by the gp41 envelope protein. The result is that the contents of the virus core are thus freed to enter the cell. The virus core is surrounded by matrix protein called p17 and is itself in the shape of a hollow cone made of another protein p24 containing the genetic material of the virus.
Being a retrovirus this genetic material is in the form of RNA (ribonucleic acid) consisting of two RNA strands. These are in turn attached to molecules of an enzyme, reverse transcriptase, which transcribes the viral RNA into DNA once virus has entered the cell. Coexisting with RNA are an integrase, a protease, a ribonuclease and other enzymes. Once in the cell the viral RNA is converted to DNA which then enters the cell nucleus. The next step is integration of viral DNA into host chromosomes. This is followed by cell proteins binding to DNA initiating transcription. Short RNA molecules then leave the nucleus and make viral regulatory proteins followed by medium length and long RNA which generate structural and enzymatic proteins. These assemble to form new viruses (replication-viral budding) (1).
Prior to 1991 the only drug available to combat HIV/AIDS was Glaxo-Wellcome's AZT (zidovudine) a nucleoside analogue which works by binding to the reverse transcriptase enzyme thereby inhibiting viral replication. Unfortunately, long term use led to the virus developing resistance against the drug by mutation. New drugs in the same class were subsequently developed including 3TC (lamivudine) (Glaxo-Wellcome), ddc (zalcitabine) (Roche), ddl (didanosine)(Bristol-Myers Squibb), d4T (stavudine) (Bristol-Myers Squibb) and recently abacavir (Glaxo-Wellcome).
1996 saw the introduction of a new class of drugs which acted at a different (and later) stage in the HIV virus' life cycle by blocking the action of the protease enzyme during viral replication. Furthermore, use of one of these with two of the class above (reverse transcriptase) gave viral loads in the blood being reduced by up to 4 log units or by a factor of ten thousand. Use of one drug alone reduces viral load by up to 2 log units or by a factor of one hundred. An effective example of this so called triple therapy would be use of AZT and 3TC (reverse transcriptase inhibitors) and indinavir (Merck Sharp and Dohme) or nelfinavir (Agouron) (protease inhibitors). Other protease inhibitors include saquinavir (Roche), ritanovir (Abbott laboratories) and amprenavir (Glaxo-Wellcome). In general, effective therapies employ two reverse transcriptase inhibitors together with one protease inhibitor.
1996 also saw the introduction of another new class of drugs known as non-nucleoside reverse transcriptase inhibitors, the first being nevirapine (Boehringer Ingelheim) followed by delavirdine. (Pharmacia Upjohn) in 1997 then efavirenz (Du Pont) in 1998.
New effective therapies also capable of reducing viral loads by up to 4 log units or by a factor of 10,000 employ a combination of nucleoside and non-nucleoside reverse transcriptase inhibitors using a total of at least three drugs.
The cost of any triple therapy per patient per year is £10,000-£15,000. (2).
The following table gives an overview of current AIDS drugs, their type or class, effectiveness in reducing viral load, total amount of drug given to patient each day in number of doses, side-effects, time for viral drug resistance to develop when used alone, and approximate cost per patient per year. (2).
The first mentioned nucleoside reverse transcriptase enzyme inhibitor zidovudine (AZT) when used by itself has subsequently been shown to provide no benefits in treating HIV-infected individuals (3) although it is effective reducing transmission from mother to baby (4).
However, it can be effective when used in conjunction with other AIDS drugs such as 3TC, another nucleoside reverse transcriptase enzyme inhibitor (5).
Additionally, the HIV virus develops viral drug resistance against AZT rather quickly (5-6 months) when used alone and even more rapidly (1 and a half months) against 3TC when used alone (2). All nucleoside revere transcriptase enzyme inhibitors can cause serious side effects ranging from myopathy to peripheral neuropathy (nerve damage). The most recent drug abacavir's side effects can be life-threatening so treatment with this drug is immediately stopped at the first signs of any adverse reactions. Also ddc is a very toxic drug. Reduction in viral loads by drugs used on their own are only moderate 50-90% and their cost is quite high (£1,200-£10,000 per patient per year) (2).
The relatively recently developed non-nucleoside reverse transcriptase enzyme inhibitor AIDS drugs can cause severe skin reaction in patients and the HIV virus can develop viral drug resistance against them very quickly in 2 months in monotherapy (one drug). In addition, cross viral drug resistance has been noted using this class of drugs. In this case drug resistance against one drug in the class can cause drug resistance against another drug of the same class (2). Again used by themselves they only reduced viral load in patients by 50-90% and are relatively expensive (£1800-£2400 per person per year) (2).
The new protease enzyme inhibitors have to be given to patients in relatively large amounts (1250-2400 mg per clay) and can give serious side effects ranging from kidney stones to hepatitis and after prolonged use patients exhibit raised levels of cholesterol and triglycerides and can cause diabetes and abnormal distribution of body fat. In addition they are expensive (£4000-£7000 per person per year) (2). They are also generally poorly absorbed and have poor bioavailability which could well be related to their low water solubility (6), (Protease Inhibitors in Patients with HIV disease by M. Barry, S. Gibbons, D. Back and F. Mulcahy in Clinical Pharmacokinetics March 32 (3) 1997 p 194) and can interact with other protease enzyme inhibitors and nucleoside/non-nucleoside enzyme inhibitors in combination therapy, giving rise to a very strict order of oral dosing which must be adhered to by the patient (7) (Pharmacokinetics and Potential Interactions amongst Antiretroviral Agents used to treat patients with HIV infection by M. Barry, F. Mulcahy, C. Merry, S. Gibbons and D. Back, Clinical Pharmacokinetics, April 36(4) 1997 p 289).
MARKETPLACE COMPARISONCOST/TOTALPATIENT/REDUCTIONAMOUNTVIRAL DRUGYEARIN VIRALDRUG/DAYRESISTANCE(PUNTS)DRUGTYPELOADin (x) dosesSIDE EFFECTS(MTHS)COMPANYZidovudinenucleoside50–90%  600 mg (2)myelosupression,5–6£7,000–£10,000(AZT)reversemyopathy, nausea,Glaxo-transcriptaseheadache, anaemiaWellcomeinhibitorLamivudinenucleoside50–90%  300 mg (2)gastrointestinal1½£7,000(3TC)reversedisturbances, hairGlaxo-transcriptaseloss,Wellcomeenzymemyelosuppression,inhibitorexacerbation ofperipheralneuropathyStavudinenucleoside50–90%  40 mg (2)peripheralgreater than 6£1,800(d4T)reverseneuropathyBristoltranscriptaseMyersenzymeSquibbinhibitorDidanosinenucleoside50–90%300–400 mgperipheralgreater than 6£2,000(ddl)reverse(1) (at night)neuropathy, nauseaBristoltranscriptasevomiting, pancreatisMyersenzymeSquibbinhibitorZalcitabinenucleoside50–90% 0.75 mg (1)very severegreater than 6£1,200(ddc)reverse(with meals)peripheral neuritisRochetranscriptaseenzymeinhibitorAbacavirnucleoside50–90%  300 mg (2)any reaction can be—£2,400reverselife-threateningGlaxo-transcriptasealways stoppedWellcomeenzymeimmediatelyinhibitorNevirapincnon-50–90%  200 mg (2)skin reaction2£1,800nucleosideBoehringerreverseIngelheimtranscriptaseenzymeinhibitorDelaviridinenon-50–90%  600 mg (3)skin reaction2£1,800nucleosidemany tabletsPharmacia-reverseUpjohntranscriptase(Agouron)enzymeinhibitorEfavirenznon-50–90%  600 mg (1)skin reaction2£2,400nucleosideDupontreversetranscriptaseenzymeinhibitorIndinavirprotease99% 2400 mg (3)hyperbilrubinaemia,6£5,000–£7,000enzymenephrolthiasis,Merck Sharpinhibitornausea, kidney& Dohmestones, dizzinessRitonavirprotease99% 1800 mg (2)diarrhoea nausea,6£5,000–£7,000(not used byenzymevomiting, hepatitis,Abottitself)inhibitorheadacheLaboratoriesSaquinavirprotease99% 1800 mg (2)loose stools, nausea,6£5,000–£7,000enzymeheadacheRocheinhibitorNelfinavirprotease99% 1250 mg (2) adiarrohea, nausea6£4,000–£5,000(Viracept)enzymelot of tablets& vomitingAgouroninhibitortotal 10(Roche)Amprenavirprotease99%a lot ofsevere rash—£7,000(can be usedenzymetabletsGlaxo-withinhibitorWellcomeRitonavir)All protease enzyme inhibitors raise patient's cholesterol, triglyceride levels and can cause diabetes, kidney stones and abnormal distribution of body fat after prolonged use.
The concentration at which an HIV-1 drug is effective is designated EC50 μm which represents when the number of cells protected from HIV injection is half the total. The antigen Agp120 assay—the virus related antigen—is related to the number of virus particles produced by measuring glycoprotein gp120 in infected cell cultures. The concentration of the drug which reduces cell growth by 50% is designated TC50 μM.
Of course the lower the EC50 concentration the better but the real criterion of effectiveness in in vitro testing on cell cultures is the Therapeutic index which is the TC50/EC50 ratio. The therapeutic index is selected so as not to damage healthy cells. Thus AZT has an EC50 of ca 0.016 μM with a TC50>1000 μM. This results in a therapeutic index of >1000/0.016=>62,500. This figure serves as a benchmark against which new potential drugs can be measured. Of course human beings and animals are more than a collection of cells and in spite of the high Therapeutic Index, AZT is quite toxic, giving rise to nerve damage and anaemia among other things (2). Nevertheless, such tests on cell cultures indicate what is a potential anti-HIV drug.
Other factors relevant to the usefulness of an anti-HIV drug are physical properties such as water-solubility for drug absorption by the patient and stability of the compound after oral intake. Thus the potentially useful drug, the anionic polysaccharide, dextran sulphate is poorly absorbed orally and degrades after oral intake before entry into the plasma (8). Another important factor is the ease of synthesis of the drug and hence drug cost which is relatively high for AZT and most other drugs produced to date which are potentially useful in combating AIDS.
International Publication No. WO9403164 describes compounds having biological activity, particularly sulfonate based calixarenes, having anti-HIV activity.
International application No. PCT/IE01/00150 relates to compounds selected from the general group of compounds disclosed in international publication no. WO 95/19974 having especially surprising activity. It relates in particular to cyclic tetrameric pyrogallol-aldehyde derivatives and to calixarene derivatives which are useful in the treatment of AIDS. In particular, International application No. PCT/IE01/00150 discloses the dodecapotassium acetate of p-bromopyrogallol P—F-phenyl tetramer (AC-1 (Example 1 in PCT/IE01/00150)).
Further studies have been flied out on synthetic routes for AC-1 which demonstrate, rather surprisingly, that a non-brominated, partially alkylated analogue of AC-1 may be more active as an anti-AIDS drug than AC-1.
International Application No. PCT/IE01/00150 and International Publication No. WO 95/19974 do not teach the formation of a partially alkylated, non-brominated analogue of AC-1. Indeed, the problems associated with the complex step of selective alkylation during the synthesis of the compound disclosed herein teach away from the formation of a partially alkylated compound. In particular, the present invention relates to a tetra-alkylated non-brominated analogue of AC-1.
The present application also relates to the use of this non-brominated, partially alkylated compound in a pharmaceutical composition for the treatment of HIV-1.
There is a need for an anti-HIV drug which brings about a reduction in viral load but without causing the development of viral drug resistance and problems of toxicity. In short, a drug is needed which when given orally gives rise to at least a M.I.C. (Minimum inhibitory concentration) of drug in the blood against HIV but at a low enough concentration so as not to give rise to adverse side effects in the patient.